The viability of eukaryotic cells relies upon the tight regulation of the initiation of protein synthesis. There is mounting evidence that this regulation involves important conformational changes within the small (40S) subunit of the ribosome. In order to probe the molecular basis and functional roles of these conformational changes in the 40S subunit, we will examine aspects of translation initiation regulation controlled by viral gene expression. Many viruses such as hepatitis C contain internal ribosome entry sites (IRESs) in their messenger RNAs that circumvent conventional initiation. These IRESs do not require the full complement of translation initiation factors to function. This enables viruses to bypass cellular pathways that inhibit translation upon viral infection. Understanding the molecular mechanisms used by viral IRESs will greatly aid in the development of antiviral strategies to combat devastating human viral diseases. The Cricket Paralysis Virus contains and IRES (CrPV IRES) that requires none of the initiation factors, and even circumvents the need for initiator tRNA. Interestingly, the CrPV IRES functions on all classes of eukaryotic ribosome, including human ribosomes. Elucidating its underlying mechanism of action will therefore reveal general features of eukaryotic ribosomes important for translation initiation. The specific aims of the proposal are the following. 1) Map conformational changes in the 40S subunit induced by the hepatitis C Virus IRES during translation initiation, and 2) map conformational changes in the 40S subunit induced by the Cricket Paralysis Virus IRES during translation initiation.